ABSTRACT
Since 1997, our IFAPA working group has developed a series of projects to evaluate new chemical and non-chemical soil disinfestation practices. During 2013–14 non-chemical (biological) alternatives have been tested with the objective of improving the biosolarization techniques, using different carbon sources and doses of organic biofumigant products under different types of plastic tarp. Throughout the growing season, the greatest production for cv. Florida Fortuna (52,672 kg/ha, by the end of May) was obtained with biosolarization using dried olive pomace (12,500 kg/ha) and virtually impermeable film; whereas biosolarization with chicken manure at 25,000 kg/ha had the lowest fruit production (42,517 kg/ha) among biosolarization treatments. Biosolarization treatments significantly reduced soil populations of M. phaseolina and Fusarium spp., and effectively suppressed nematode infestation.
Introduction
Since 1997 a series of field trials with chemical and non-chemical alternatives have been conducted in conventional crop systems in the coastal area of Huelva (Spain), the main strawberry production area in Europe (Medina-Minguez et al., Citation2012). So far, more than 20 alternative fumigants have been evaluated in this series of experiments, such as: 1,3-dichloropropene + chloropicrin, chloropicrin alone, metam sodium, dimethyl disulphide, or dazomet among others (López Aranda et al., Citation2009a, Citation2009b). However, the policy trends in developed countries are towards restricting use of chemical fumigants. For this reason, alternative control techniques are needed.
Non-fumigant soil disinfestation for strawberry production, such as steam, soil solarization, soil-less cultivation, biofumigation, and others, are still considered risky and/or uneconomical to be used alone as global alternatives to chemical fumigants (Ajwa et al., Citation2003). Therefore, they are not yet available for a generalized use in any significant strawberry growing areas. Despite that, in some countries the research on soil disinfestation alternatives has been intensive, mainly in the United States (Rosskopf et al., Citation2015; Shennan et al., Citation2014) and Spain (Domínguez et al., Citation2014; López-Aranda et al., Citation2009a; Medina-Mínguez et al., Citation2012).
Research on biofumigation in combination with solarization (biosolarization) is relatively recent and scarce. Most of the studies focused on biosolarization as a remediation tool for soils polluted with pesticides that likely relies on an increase in soil temperature for enhanced pesticide degradation (Fenoll et al., Citation2010). Additionally, biosolarization was tested for control of soil-borne fungi associated with yield decline, such as Phytophthora capsici or Fusarium spp., in temperate climate regions (Martínez et al., Citation2011; Núñez-Zofio et al., Citation2011). However, this technique could be considered a viable alternative to chemical soil fumigation only if it maintains comparable strawberry yields over time.
The aim of this work was to further develop and improve the biosolarization techniques, while complying with the regulatory standards for nitrate nitrogen in groundwater and surface water runoff (Council Directive 91/676/EEC concerning the protection of waters against pollution caused by nitrates from agricultural sources—“EU Nitrates Directive”).
Material and methods
During the 2013–14 growing seasons, a field trial with non-chemical alternatives has been conducted in an experimental field at Moguer, Huelva. The field trial was arranged in complete randomized blocks (four replications, 20 m2 plots) with seven different biosolarization treatments and an untreated control (). Biofumigant treatments were applied during the 2nd week of July and soil solarization was conducted from mid-July to mid-August using conventional (HDPE) or virtually impermeable film (VIF) transparent film (42.5 and 25 microns thick, respectively) (). Also, C, N, and moisture content of fresh chicken manure and dried olive pomace used as the main biofumigant sources are presented in . The method for applying Trichoderma in treatment D was: Trichoderma Tusal (Trichoderma asperellum + Trichoderma atroviride) by immersion of the transplants before planting (2 kg/m3 of water + amino acids 2 l/m3 of water), and six applications by drip irrigation system during the cultivation cycle (5 Dec. 2013; 18 Dec. 2013; 17 Jan. 2014; 17 Feb. 2014; 21 Mar. 2014; 14 Apr. 2014).
Table 1. Treatments applied.
‘Florida Fortuna’ (marketed as ‘Florida Radiance’ in the U.S.) plants were planted at mid-October and were grown following standard production and IPM practices under high plastic tunnels until late May.
Sanitary status of plants was determined by sampling 50 plants before planting. To detect fungal infections, crowns and 5-cm-long root segments were surface disinfected, plated on potato dextrose agar, and cultivated at 25 °C for 7 days in the dark. To detect nematode infections, plant roots and leaves were cut into small pieces and processed by the sugar centrifugation method (Nombela and Bello, Citation1983).
Soil populations of Macrophomina phaseolina and Fusarium spp. were estimated in each plot before (early July) and after (October) treatments as CFU per gram of dry soil to evaluate the effectiveness of the treatments. Before treatments, five samples were taken in a zig-zagged pattern in each plot and mixed to make a composite sample. After treatments and at the end of the season, three samples were taken from the central bed of each plot and mixed to make a composite sample. Soil samples were taken from the first 20 cm depth using a vertical calibrated drill. Soil samples were air dried and processed as follows. One or 7 grams of soil per sample taken in July or in October, respectively, were suspended in 150 ml of water agar (0.1%). Aliquots of 1 ml were spread on petri dishes with V-8 semi-selective media to quantify the presence of Fusarium spp. (Komada, Citation1975). The M. phaseolina isolations were carried out following a modification of the technique described by Papavizas and Klag (Citation1975) (Chamorro et al., Citation2015). Data were transformed (square root) prior to analysis. Means were compared using an LSD protected test at the 5% significance level (Statistix 8.0; Analytical Software, Ltd., La Jolla, CA, USA).
Plant survival percentages were determined weekly, beginning at planting and continuing until the end of the growing season. Dead plants from each subplot were collected until mid-May for pathogen diagnosis. Plants were washed under running tap water, sectioned into roots and crown parts, surface-sterilized in 1% aqueous solution of sodium hypochlorite for 2 min, rinsed twice in sterile distilled water, and dried in a laminar flow cabinet. Pieces of root and crown were then separately placed on non-selective potato dextrose agar medium (PDA). Cultures isolated were inspected microscopically for identification. The incidence of mortality was expressed as percentage of dead plants. Data were transformed (arcsine square root) prior to analysis. Means were compared using the LSD protected test at the 5% significance level (Statistix 8.0; Analytical Software, Ltd., La Jolla, CA, USA).
Ten randomly selected plants from each replication were observed throughout the complete growing season; plant diameter (as a measure of plant vigor) was determined by taking one-dimensional measurements across the plant leaf canopy. The incidence of nematodes in the plants was assessed at the end of the season (early-May) on the same plants selected for plant vigor. The Gall Index was recorded in a 0–4 rating scale (0 = absence of symptoms and 4 = all roots infested; Barker, Citation1985). Roots from the five selected plants from each replication were joined in one single sample of 10 g, and sedentary forms of Meloidogyne hapla were extracted and quantified by the sugar centrifugation method (Nombela and Bello, Citation1983).
Individual fruit weight was estimated by weighting 20 randomly selected marketable fruits at every third harvest. Extra-early (by the end of February), early (by the end of March), and total yield (by the end of May) data from the total number of plants per plot was obtained at least once each week throughout the production season (from mid-January to the end of May). Fruit was graded into two commercial categories using the European regulations criteria for fresh fruit: first and second category (Commission Regulation CE 843/2002 laying down the marketing standard for strawberry and amending Regulation EEC 898/1987). First and second categories were considered marketable. Fruit in the first category are healthy, well-shaped with a weight above 14–15 g per fruit. Fruit in the second category are healthy fruit, well-shaped and with a weight below 14–15 g per fruit (and/or healthy fruit lightly misshapen of size above 14–15 g/fruit). Non-marketable (culls) fruit were rejected without recording (less than 2–3% of harvested fruit, data not shown).
Data were submitted to analysis of variance and treatment means were compared with Fischer’s protected least significant difference test at the 5% significance level (Statistix v. 8.0, Analytical Software, Tallahassee, FL, USA).
Results and discussion
Fungi
In soil samples taken before treatments, the average number of M. phaseolina propagules per gram of soil in individual plots ranged from 0 to 24 CFU. While Fusarium spp. levels in soil prior to treatment application were similar, M. phaseolina populations in soil (CFU/g) were significantly different (P = 0.05) among treatments (). Biosolarization treatments significantly reduced the soil populations of M. phaseolina and Fusarium spp. compared to the UC ().
Table 2. Biosolarization treatment effect on Macrophomina phaseolina and Fusarium spp. populations (CFU/g of dry soil) in strawberry soils.
Trichoderma spp., Fusarium spp., and Rhizoctonia sp. were isolated from the bare root strawberry transplants before planting. Wilting of plants first appeared at the end of November. Phoma spp. and Fusarium spp. were isolated from symptomatic crowns, whereas Cylindrocarpon sp., Rhizoctonia sp., and Fusarium spp. (casual agents of strawberry black root rot) were isolated from roots. At the end of February, plants with symptoms of charcoal rot were first detected and M. phaseolina was consistently isolated from crowns and roots of symptomatic plants. Biosolarization treatments significantly reduced plant mortality and charcoal rot incidence ().
Table 3. Effect of soil disinfestation treatments on plant mortality and incidence of charcoal rot (caused by Macrophomina phaseolina) during the 2013–14 strawberry season (means represent accumulated plant mortality until the end of each season—mid May).
Nematodes
All treatments reduced the severity of the plant decline caused by M. hapla, as estimated by the galling index and nematode densities within the roots (both significantly reduced in all treatments when compared to controls (). There were no significant differences among biofumigant treatments neither in galling index nor in nematode densities (). All biosolarization treatments were effective in suppressing nematode symptoms and reducing final nematode densities at the end of the cropping season, but at this stage there were no differences in efficacy between them.
Table 4. Meloidogyne hapla densities in strawberry roots as influenced by biofumigant treatments.
Plant vigor
At early season (end of December), the diameter of the plants did not differ significantly among treatments (22.4–23.3 cm) with the exception of the untreated control that always remained the least vigorous treatment with an average canopy diameter of 20.7. A similar trend continued throughout the season and from late April until the end of the season plant diameters remained at 34.6–36.9 cm for all biofumigant treatments and 32.7 cm for untreated control.
Percentage of dead plants
Throughout the whole season, the percentage of dead plants was high. The mortality increased until February, particularly in the biosolarization treatment with fresh chicken manure (12,500 kg/ha) and Trichoderma reaching 10.3% in November and 12.5% by January. In March, no significant increase of dead plants was observed, but at the end of April and May, again a considerable increase was detected, due probably to the temperature rise during these months (data not shown). During this period, the untreated control and the biosolarization with fresh chicken manure (25,000 kg/ha) had the highest plant mortality, while biosolarization with dried olive pomace (12,500 kg/ha) and VIF film had the lowest plant mortality.
Fruit yield
Until the 28th of February, the marketable first class yield was not affected by the treatment (). However, the treatment with the highest yield was the biosolarization with dried olive pomace (12,500 kg/ha) and VIF film (treatment H; 9.749 kg/ha). At the end of March, again, no significant differences among biosolarization treatments were observed, although it was significantly lower in the untreated control (). At the end of the cropping season (end of May), the untreated control continued to have the lowest yield, but there were also differences among of the biosolarization treatments. Consistent with previous harvest periods, biosolarization with dried olive pomace and VIF film, again produced the highest yield, while biosolarization with fresh chicken manure (25,000 kg/ha), had the lowest yield ().
Table 5. First-class fruit yields, percentage of second-class fruit, and fruit weight as influenced by biofumigant treatments.
Percentage of second class fruit
The untreated control had a significantly higher percentage of second class fruit, compared to biofumigant treatments, which had a similar fraction of second class fruit ().
Fruit weight
Fruit weight in all biofumigant treatments was greater than in the untreated control except the biosolarization with fresh chicken manure (25,000 kg/ha) that had fruit weights similar to the untreated control.
Conclusions
The study showed that all biosolarization treatments can be effective in reducing soil populations of Fusarium spp. and Macrophomina phaseolina, and the plant mortality due to these pathogens. In addition, all treatments were effective in suppressing nematode symptoms and reducing final nematode densities.
Dried olive pomace improved the productive results of the biosolarization treatments with chicken manure. On the other hand, the use of virtually impermeable film (VIF) did not involve any improvement. Likewise, neither the application of Trichoderma nor the application of glicerin improved the biosolarization treatment with fresh chicken manure (12,500 kg/ha).
Funding
This research was supported by the projects: TRA.TRA201300.6—“Mejora de la competitividad en el cultivo de la fresa mediante experimentación, transferencia y formación”; the complementary project PP.AVA.AVA201301.6; and FEDER and FSE funds.
Additional information
Funding
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